Breast cancer is one of the common malignancies of women, and its incidence is in the first place among female cancer patients. Currently the primary treatment procedures for breast cancer include surgery, radiotherapy, chemotherapy, and hormone therapy, etc. Chemotherapy is always playing an important role in the comprehensive treatment of breast cancer due to the sensitivity of breast cancer to anti-cancer drugs Traditional drugs for chemotherapy in breast cancer mainly include doxorubicin, cyclophosphamide, 5-fluorouracil, etc. Although these drugs have been widely used in the treatment of breast cancer, their therapeutic efficacy is limited due to the toxicity and resistance of patients. In recent years, targeted therapy is used clinically, with the primary drugs like Herceptin, tyrosine kinase inhibitor, lapatinib, pertuzumab monoclonal antibody, bevacizumab, flavopiridol, etc. However, due to patient's tolerance, physical properties of drugs like instability and solubility and the targets, targeted therapy only applies to a subgroup of patients. With the advances in bacterial- and viral-based gene therapy and genetic engineering technology, mounting studies have focused on bacterial treatment of tumors since the middle 1990s. Results have shown that Salmonella typhimurium can inhibit the growth of tumor cells in mice in a targeted and efficient manner.
Salmonella is a group of Gram-negative, invasive intracellular facultative anaerobes parasitized in human and animal intestinal tracts. VNP20009 is an attenuated Salmonella typhimurium strain with the deletion of msb B and pur I genes. It is genetically stable and sensitive to antibiotics. The msb B protein is necessary for the lipid acylation to endotoxin, and the lipid acylation at A-terminal cannot be achieved when deleted, lowering the toxicity. The pur I protein is involved in purine metabolism, deletion of this gene leads to dependence of exogenous adenine when culturing the bacteria. These gene manipulations in VNP20009 also lower the production of tumor necrosis factor (TNF), thereby reducing the inflammatory response. Consequently, the low pathogenicity improves the safety of its clinical usage. VNP20009 has been widely used in cancer research, which can influence the growth of a variety of solid tumor models of mice, including melanoma, lung cancer, colon cancer, breast cancer, renal cancer and prostate cancer. VNP20009, as a vector of gene therapy, has the ability to accumulate in the tumor site in a highly targeted fashion. Researchers have found in the mouse models carrying a variety of solid tumors that the quantity of VNP20009 in tumors is 200-1000 times as high as that in non-cancerous major organs, such as the liver. It uses a more complex set of mechanisms to target tumors. VNP20009 can preferentially accumulate and multiply under the hypoxic and necrotic conditions in the tumor tissue. At the same time, the bacteria multiply significantly faster in the tumor tissues than in the normal tissues, making it possible for the attenuated Salmonella to be a new type of anti-tumor agent and the vector of targeted gene therapy. Potential mechanisms for the effect of a slow tumor growth by VNP20009 may include the follows: 1) Breakdown of nutrients necessary for tumor growth by the bacteria, e.g., the enzymes produced by bacteria such as asparaginase, can deplete essential amino acids for tumor growth; 2) Stimulation of local toxin secretion or tumor necrosis factor α to tumor microenvironment can negatively influence the tumor angiogenesis. In addition, the non-specific inflammatory reaction at the bacterial growth site can activate anti-tumor T cells. Studies have shown that although attenuated Salmonella VNP2009 is an ideal carrier for gene therapy which can be applied safely with high allowable dose, its application independently has no strong anti-tumor effect and a further combination with other drugs is needed.
Tumor cells require adequate nutrition in order to maintain its high rate of reproduction. In addition to carbohydrates, the need for methionine (Met), glutamine, and arginine is particularly high. Previous studies have established that Met-dependency is a common feature of most tumor cells, such as breast cancer, lung cancer, colon cancer, kidney cancer, bladder cancer, melanoma, glioma, etc. High Met-dependency does not exist in normal cells. Both in vivo and in vitro experiments have confirmed that dietary intervention with methionine deficiency can delay the proliferation of tumor cells. However, long-term deficiency of Met can cause malnutrition, metabolic disorders, and aggravate tumor growth due to a long-term DNA hypomethylation. Thus, by specifically degrading Met to methylselenol, a-ketobutyrate and ammonia through L-methioninase and lowering the level of methionine in vivo, we will be able to effectively inhibit the growth of tumor cells or even degrade them. Experiments in animal models have confirmed that intraperitoneal injection of methioninase can inhibit the growth of Yoshida sarcoma and lung tumor in nude mice. In previous clinical trials, four patients with breast cancer, lung cancer, kidney cancer and lymphoma received methioninase injection once every 24 h. Methioninase could significantly reduce the methionine content in plasma. However, since methioninase is not natively expressed in mammalians, exogenous administration often causes the immunological response.